Japan Association Of Childhood Leukemia Study Research Articles

Differential impact of asparaginase discontinuation on outcomes of children with T-cell acute lymphoblastic leukemia and T-cell lymphoblastic lymphoma.

Asparaginase is essential for treating T-cell acute lymphoblastic leukemia (T-ALL). Despite the ongoing debate on whether T-ALL and T-cell lymphoblastic lymphoma (T-LBL) are the same disease entity or two distinct diseases, patients with T-LBL often receive the same or similar treatment protocols as those with T-ALL. The outcomes of patients with or without L-asparaginase discontinuation were retrospectively analyzed among four national protocols: Japan Association of Childhood Leukemia Study (JACLS) ALL-02 and ALL-97 for T-ALL and Japanese Pediatric Leukemia/Lymphoma Study Group ALB-NHL03 and JACLS NHL-98 for T-LBL. The hazard ratio (HR) was calculated with the Cox regression model by considering L-asparaginase discontinuation as a time-dependent variable. In total, 199 patients with T-ALL, and 133 patients with T-LBL were included. L-asparaginase discontinuation compromised event-free survival (EFS) of T-ALL patients (ALL-02: HR 3.32, 95% confidence interval [CI] 1.40-7.90; ALL-97: HR 3.39, 95%CI 1.19-9.67). Conversely, EFS compromise was not detected among T-LBL patients (ALB-NHL03: HR 1.39, 95%CI 0.41-4.68; NHL-98: HR 0.92, 95%CI 0.11-7.60). The effects of L-asparaginase discontinuation differed between T-ALL and T-LBL. We assume that the differential impact results from (1) the inherent differential response to L-asparaginase between them and/or (2) a less stringent assessment of early treatment response in T-LBL than in T-ALL. Given the poor salvage rate of refractory or relapsed T-ALL and T-LBL, optimization of the frontline therapy is critical, and the current study provides a new suggestion for further treatment modifications. However, larger studies in contemporary intensified treatment protocols are required.

JACLS ALL-02 SR protocol reduced-intensity chemotherapy produces excellent outcomes in patients with low-risk childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. As overall cure rates of childhood ALL have improved, reduction of overall treatment intensity while still ensuring excellent outcomes is imperative for low-risk patients. We report the outcomes of patients treated following the standard-risk protocol from the prospective Japan Association of Childhood Leukemia Study (JACLS) ALL-02 study, which was conducted between 2002 and 2008 for patients with newly diagnosed ALL aged 1-18years. Of 1138 patients with B-cell precursor ALL, 388 (34.1%) were allocated to this protocol. Excellent outcomes were achieved despite the overall treatment intensity being lower than that of most contemporary protocols: 4years event-free survival (EFS) was 92.3% and 4years overall survival 98.2%. Patients with high hyperdiploidy (HHD) involving triple trisomy (trisomy of chromosomes 4, 10, and 17) or ETV6-RUNX1 had even better outcomes (4years EFS 97.6% and 100%, respectively). Unique characteristics of this protocol include a selection of low-risk patients with a low initial WBC count and good early treatment response and reduction of cumulative doses of chemotherapeutic agents while maintaining dose density. In Japan, we are currently investigating the feasibility of this protocol while incorporating minimal residual disease into the patient stratification strategy.

The Outcome of Childhood B-Cell Precursor ALL with ETV6-RUNX1 Treated with Japan Association of Childhood Leukemia Study (JACLS) ALL-02 Trial

Background: It is well known that ETV6-RUNX1 positive pediatric B cell precursor acute lymphoblastic leukemia (B-ALL) shows excellent outcome when they are treated with contemporary chemotherapeutic regimens. However, the prognosis of ETV6-RUNX1 positive B-ALL wasn't determined in Japanese pediatric cohort which was uniformly treated. Japan Association of Childhood Leukemia Study (JACLS) ALL-02 protocol achieved 83.7±1.1% of 4-year event free survival of pediatric B-ALL (Hasegawa et al. 58th ASH annual meeting) which was comparable to that in major recent pediatric ALL clinical trials, although risk stratification based on minimal residual disease (MRD) was not employed. Herein, we report the outcome and analyze the prognostic factors of ETV6-RUNX1 positive B-ALL treated with JACLS ALL-02 protocol.Patients and treatment: Between April 2002 and March 2008, newly diagnosed de novo ETV6-RUNX1 positive ALL with 1-18 years of age were enrolled to JACLS ALL-02 trial. Patients were stratified into 3 groups: standard-risk (SR), high-risk (HR), extremely high-risk (ER), based on initial prednisolone (PSL) response and the modified National Cancer Institute(NCI) workshop criteria. Prednisolone poor response (PPR) was determined after 7 days of monotherapy with prednisone and one intrathecal dose of methotrexate. PSL good responders (PGR; < 1,000 blasts/μL) were divided into SR and HR according to the modified NCI workshop criteria by WBC 10K and age 10, and received conventional therapy. BCP-ALL with PPR (≥ 1,000 blasts/μL) were assigned to ER and received intensified post-induction therapy. Estimation of event free survival (EFS) and overall survival (OS) was performed using the Kaplan-Meier method and the differences were compared using the log-rank test. Multivariate analysis was performed using a Cox regression model.Results: 205 newly diagnosed ETV6-RUNX1 positive ALL patients were enrolled in JACLS ALL-02 clinical trial, which included 108 patients in SR group (52.7%), 89 in HR group (43.3%) and 8 in ER group (3.9%). The median age at diagnosis was 4.3 years (range, 1.0 to 15.0 years) and the median WBC count was 6,800/μL (range, 370 to 384,700 /μL). The median follow-up time was 6 year and 6 months. 4y/8y EFS and OS (±SE) are 92.5±2.6/ 89.8±2.5% and 96.5±1.5/ 95.5±1.6%, respectively. 4y / 8y EFS of the patients of NCI-SR (n=167) and NCI-HR (n=38) are 95.6±1.6% / 91.3±2.7% and 89.5±5.0% / 83.0±6.4%, respectively (log rank p=0.04). 4y / 8y OS of the patients with PSL good response (n=197) was 97.9±1.1% / 97.0±1.3%, while those of the patients with PPR (n=8) was 87.5±11.7% / 58.3±18.6% (log rank p<0.001). Eighteen of 205 (8.8%) patients experienced relapse. According to BFM risk stratification of relapsed ALL (S1 to S4), 13 of 18 patients were in S2 and ten of them were rescued by salvage therapy. However, 5 patients in S3 or S4 group died of disease progression. Although two patients showing PPR belonged to S2, all of them died of disease progression. Multivariate analysis identifies NCI-HR (HR=2.92, p=0.05) as a predictor of poor prognosis in EFS, and PPR (HR=12.0, p<0.01) as predictor of poor prognosis in OS.Discussion: Although JACLS ALL-02 protocol didn't employ MRD based risk stratification, good outcome of the 4y EFS and OS (92.5±2.6 % and 96.5±1.5 %) was achieved for ETV6-RUNX1 positive B-ALL patients. Because NCI-SR and PGR were predictors of excellent outcome for ETV6-RUNX1 positive B-ALL, less intensive treatment should be explored for this group. On the other hand, NCI-HR and PPR indicated poor outcome. We hope that MRD based monitoring might improve outcome of this subgroup in our next clinical trial. DisclosuresNo relevant conflicts of interest to declare.

The Epigenetic and Genetic Landscapes of Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL)

IntroductionAlthough genetic basis of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has been well characterized and we reported recurrent SPI1 fusion cases exhibiting uniformly dismal outcome, no comprehensive studies have yet explored the epigenetic profiles and their potential contribution to clinicopathological features of T-ALL.MethodsA total of 79 cases with pediatric T-ALL samples from Tokyo Children's Cancer Study Group (TCCSG) and Japan Association of childhood Leukemia Study (JACLS) including 7 cases of SPI1 fusions were analyzed in this study. To describe epigenetic landscape of T-ALL, we performed methylation array analysis using Illumina HumanMethylation EPIC array, and conducted combined analysis of methylation data with our previous data of whole transcriptome sequencing (WTS) and targeted-capture sequencing for 158 ALL-related genes.ResultsAfter normalization, 939 probes were selected to identify the most variable methylated probes, when a standard deviation of the beta-values across the samples was above 0.38. Unsupervised consensus clustering clearly identified 4 distinct sample clusters. Combined analyses with WTS data revealed that these 4 clusters were characterized by TAL1 fusions/high expression with intermediate methylation (M1 cluster; n=39), high TLX/HOX expression with intermediate methylation (M2 cluster; n=20), high TLX//HOX expression with high methylation (M3 cluster; n=11), and SPI1 fusions/high expression with low methylation (M4 cluster; n=9), respectively. Interestingly, these methylation clusters were well correlated with genetic features, expression status, and clinical outcomes. In M1 cluster, PTEN abnormalities and UPS7 mutations were especially enriched, and these aberrations were observed mutually exclusive. Furthermore, promoter regions in TAL1 and its target ALDH1A2, important for retinoic acid synthesis, were significantly hypomethylated and highly expressed in this cluster. In M2 cluster, genes related to chromatin remodeling and JAK-STAT were frequently mutated. PRR5L and DEPTOR, both related to AKT1 activation, were differentially methylated and expressed, suggesting an important role of PI3K-AKT1 pathway. M3 cluster was characterized by frequent mutations of epigenetic regulators related genes, such as PHF6, EZH2, and DNM2 . In contrast, mutations in JAK-STAT and PI3K-AKT1 pathways were less frequent. Since the methylation profile of M3 cluster was similar to normal T cells, abnormal DNA methylation status may be not crucial for the leukemogenesis in this group. Intriguingly, all SPI1 fusion cases were classified into M4 cluster, and remaining 2 cases without SPI1 fusions in this cluster showed very high SPI1 expression. Importantly, M4 cluster was characterized by high expression of RASGRP4, a family of Ras guanine nucleotide exchange factors, and low methylation of RASGRP4 promoter. In addition, FGFR1 expression was uniformly repressed and abnormalities of CDKN2A (89%) and KRAS/NRAS (44%) were frequently observed in this group. Furthermore, it should be noted that patients in M4 cluster showed significantly poor prognosis compared to other clusters; eight out of nine cases were died (Log-rank p =4.4×10-7). SPI1 fusion cases exhibited not only unique cytological and gene expression profiles but also distinctive methylation status.ConclusionsBased on DNA methylation profiles, pediatric T-ALL is clustered into 4 distinct subtypes, which exhibited remarkable correlation with genetic signatures, expression features, and clinical outcomes. PI3K-AKT pathway may be essential in M1/M2 cluster, whereas genetic abnormalities of epigenetic regulators were characteristics in M2/M3 clusters. In addition, the involvement of JAK-STAT pathway would be unique in M2 cluster. Given that cases with M4 cluster showed a catastrophic prognosis and very high expression of RASGRP4 with high frequency of RAS mutation, our findings implicate that targeting therapy of RAS may improve the prognosis of the patients in this group. Although our cohort is limited, our results suggested that the biological phenotype of T-ALL is mediated by both genetic and epigenetic regulations, and explorations for aberrant DNA methylation along with genetic alterations might be helpful for a new therapeutic strategy for T-ALL. DisclosuresNo relevant conflicts of interest to declare.

Outcome of Hematopoietic Stem Cell Transplantation in the Patients with Pediatric Acute Lymphoblastic Leukemia Enrolled in the Japan Association of Childhood Leukemia Study (JACLS) ALL-02

Background: Though the outcome of patients with acute lymphoblastic leukemia (ALL) has been greatly improved, some high-risk patients still need hematopoietic stem cell transplantation (HSCT). In this study, we evaluated clinical characteristics of children with ALL who were treated according to Japan Association of Childhood Leukemia Study (JACLS) ALL-02 trial and received HSCT to determine prognostic factors for the outcome of HSCT. Methods: Between April 1, 2002 and March 31, 2008, 1,252 patients aged 1-18 years with newly diagnosed ALL were enrolled in JACLS ALL-02 trial. Ph+ALL, mature B-ALL, and NK-leukemia were excluded. Of all the 1,252 patients, 211 (16.8%) patients were reported to receive HSCT, of which 206 patients with adequate information of 1st HSCT were subjected to further analysis. In this study, HSCT in 1st complete remission (CR) in JACLS ALL-02 trial was indicated for the patients with extremely high risk (ER: BCP-ALL with PPR and/or evidence of t(4;11) (or KMT2A-AFF1 positive), hypodiploidy (≤ 44) and/or acute mixed lineage leukemia/ acute unclassified leukemia). As a precaution, in ER patients, the timing of HSCT differs depending on whether they have a matched related donor. Results: Of all the 206 patients, 83 patients received HSCT in first CR (CR1), 68 patients in CR2, 54 patients in non-CR and 1 patient in induction failure (IF). 5-year overall survival (5y-OS) for 206 patients was 50% (95% CI 42.7-56.8). In detail, 73.8% (95% CI 62.6-82.1) for CR1 patients, 49.4% (95% CI 35.9-61.5) for CR2 patients, and 14.8% (95% CI 6.9-25.5) for non-CR patients, respectively. In univariate analysis, JACLS risk classification, disease status at HSCT (CR or non-CR or IF), stem cell source were significant prognostic factors. In multivariate analysis, only disease status retained as prognostic factor (p&lt;0.01, HR=1.77). For CR1 patients, stem cell source was significant prognostic factor on multivariate analysis (5y-OS = 41.7%, 66.7%, and 79.3% for peripheral blood, cord blood, bone marrow; p = 0.023). When we focused on immunophenotype, stem cell source retained their effect only for patients with T-ALL. For CR2 patients, central nerves system involvement at diagnosis (p=0.02, HR=7.2), S classification (p=0.03, HR=1.2), and NCI risk (p=0.04, HR=2.5) were significant prognostic factors on multivariate analysis. S classification retained its prognostic impact only for patients with BCP-ALL (p=0.01, HR=1.5). For non-CR patients, NCI or JACLS risk classification at first diagnosis, S classification, frequency of relapse before HSCT, stem cell source, and conditioning for HSCT did not affect treatment outcome. Interestingly, non-CR patients with high-hyperdiploid (HHD) (n=5) had a significantly better 5y-OS (80%) than the other subtypes. In multivariate analysis, age at diagnosis (&lt;10 years, p=0.04, HR=2.0)) and HHD (p=0.006, HR=1.74) contribute to the better outcome of HSCT in non-CR. Conclusion: The current study showed that the prognostic factors for HSCT varied according to the disease status or disease immunophenotype. Also, it is noteworthy that HHD patients may be rescued even in non-CR status by HSCT. Disclosures No relevant conflicts of interest to declare.

High rates of ovarian function preservation after hematopoietic cell transplantation with melphalan-based reduced intensity conditioning for pediatric acute leukemia: an analysis from the Japan Association of Childhood Leukemia Study (JACLS).

Women are at high risk of hypergonadotropic hypogonadism after hematopoietic cell transplantation (HCT). Hypogonadism is universal after irradiation or busulfan. We hypothesized that reduced intensity conditioning (RIC) might protect ovarian function after HCT. We retrospectively reviewed data from patients with acute leukemia treated according to the Japan Association of Childhood Leukemia Study and nationwide multicenter study protocol. We selected 11 female patients with acute leukemia who received first HCT with RIC, had survived for three or more years after HCT, and were aged ≥ 12years at the last follow-up visit. Median age at diagnosis, HCT, and last visit were 8, 10, and 17years. Six patients received HLA-matched bone marrow (BM), two HLA-mismatched BM, and three cord blood. Melphalan was used as conditioning regimen in all patients. At the last visit, six of seven post-pubertal patients at transplantation recovered menstruation, and four of four patients who underwent transplantation at the pre-pubertal began menstruation. Height z scores showed no significant reduction between pre-transplant and post-transplant. No patients received growth hormone treatment. Only one recipient displayed subclinical hypothyroidism. Melphalan-based RIC may be an encouraging option for patients with acute leukemia to avoid ovarian and endocrine dysfunction after HCT.

Coagulation and Fibrinolysis Imbalance Demonstrated By Novel Simultaneous Thrombin and Plasmin Generation Assay during L-Asparaginase Treatment Phase of Induction Therapy in Pediatric Acute Lymphoblastic Leukemia

Introduction: L-asparaginase (L-Asp) is one of the risk factor of thromboembolism during ALL chemotherapy. Although the pathogenesis has been still unclarified, L-Asp may have profound effects on hepatic synthesis of pro-, anti-coagulant and fibrinolytic factors. In addition, recent studies demonstrated that the age of over 10-years might be a risk factor for this L-Asp associated coagulopathy. In this study, we hypothesized that change of balance between coagulation and fibrinolysis contributes to hyper-coagulation condition during chemotherapy including L-Asp. In order to clarify our hypothesis, we investigated the global coagulation and fibrinolytic function during the ALL induction therapy with simultaneous thrombin and plasmin generation assay (T/P-GA). Patients: Seventy-two pediatric patients aged 1.0 to 15.2 years (43 males and 29 females) with newly diagnosed ALL were enrolled from Aug. 2014 to Mar. 2018 at four hospitals in Japan. All patients and their families had no thrombotic predisposition. Fifty-five cases (76.4%) (BCP-ALL; n=47, T-ALL; n=7, MPAL; n=1) received BFM-95 oriented protocol consisting of a total of 8 doses of E.coli L-Asp 5,000 U/m2, whilst the others (BCP-ALL; n=13, T-ALL; n=2, MPAL; n=1, Ph-ALL; n=1) received Japan Association of childhood Leukemia Study (JACLS)-ALL02-protcol consisting of a total of 6 doses of E.coli L-Asp 6,000 U/m2. Fifty-two cases (72.2%) were categorized to younger group (1-10 years; median, 4.3 years) and the other cases to older group (>10 years; median, 12.6 years). The percentage of patients of each category were similar in the two treatment protocols (p=0.764). Methods: The global functions of coagulation and fibrinolysis were evaluated using T/P-GA [Matsumoto et al. TH 2013]. This clotting was initiated by the mixture of recombinant human tissue factor (Innovin®, f.c. 1 pM), phospholipid vesicles (f.c. 4 μM) and tissue-type plasminogen activator (f.c. 3.2 nM). Thrombin and plasmin generation were monitored simultaneously using thrombin- and plasmin-specific fluorogenic substrate (Z-Gly-Gly-Arg-AMC and BOC-Glu-Lys-Lys-MAC, respectively) in separate microtiter wells. The first derivatives (velocity) of thrombin and plasmin generation were utilized to derive the parameters, lag time (LT), endogenous potential (EP), peak levels (peak), and time to peak (ttPeak). EP of thrombin generation (T-EP) and plasmin-peak (P-peak) were selected as parameters for evaluation in this study. A ratio of T-EP and P-peak of patients' plasmas to those of control normal plasma were calculated. The conventional laboratory markers of coagulation and fibrinolysis were also monitored by fibrinogen (Fbg), fibrin-Fbg degradation products (FDP), antithrombin (AT), thrombin-AT complex and plasmin-α2 plasmin inhibitor complex. Plasmas were collected at T0; pre-phase of L-Asp, T1; intermittent phase of L-Asp, T2; post-phase of L-Asp, and T3; post induction phase. Results: None of the cases developed L-Asp associated coagulopathy, and two cases (2.8%) developed induction failure. Thirteen cases (18.1%) received fresh frozen plasma transfusion for low Fbg level, whilst 52 (72.2%) cases received AT supplement for low AT level. Fbg showed a median of 165, 99.0, 97.0 and 316 mg/dl at T0, T1, T2 and T3, respectively, and AT showed a median of 143, 80.9, 80.0 and 105%, respectively, whilst the value of other conventional markers remained within reference value. T-EP revealed a median of 1,134, 1,135, 1,221, 1,277 and 1,102 nM, whilst P-peak showed a median of 5.33, 3.75, 3.63, 4.91 and 5.51 nM for T0, T1, T2, T3 and control plasma, respectively, indicating the elevated T-EP ratios and reduced P-peak ratios (Fig. 1). T-EP ratios showed significantly higher at T2 and T3 than at T0 and T1 (p<0.05), whilst P-peak ratios showed significantly lower at T1 and T2 than at T0 and T3 (p<0.01) (median ratios of T-EP/P-peak; T0; 1.06/0.97, T1; 1.04/0.65, T2; 1.12/0.65, T3; 1.17/0.87, respectively). P-peak ratios of the older group showed significantly lower than the younger group at T1 (median; 0.58 vs 0.69, p=0.016). Conclusion: The results from T/P-GA showed that their hemostatic dynamics appear likely to shift to hyper-coagulation and hypo-fibrinolysis at L-Asp treatment phase. These results suggest that the imbalance of coagulation and fibrinolysis might cause thrombotic complication during the L-Asp treatment for ALL, especially in patients aged 10-years older. [Display omitted] DisclosuresNogami:Chugai Pharmaceutical Co., Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Anti-FIXa/X bispecific antibodies , Research Funding, Speakers Bureau.

Distribution and Clinical Features of NOTCH1 Signaling Activating Alterations in Pediatric T-Cell Acute Lymphoblastic Leukemia (T-ALL)

IntroductionNOTCH1 and FBXW7 alterations leading to aberrant activation of NOTCH1 signaling, classified into two patterns; ligand-independent activation (LIA) and impaired degradation (ID) of NOTCH1. In general, activation of NOTCH1 axis is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL), though comprehensive studies regarding subclonal mutations inducing NOTCH1 activation are still elusive. In the present study, we explored the clinicopathological relevance of NOTCH1/FBXW7 aberrations considering subclonal alterations.MethodsA total of 176 cases with pediatric T-ALL were enrolled in this study. We reanalyzed our previous data of targeted-capture sequencing (n=176) for 158 ALL-related genes/regions and combined with previous expression profiling data based on whole transcriptome sequencing (WTS; n=121). We defined as a subclonal mutation when variant allele frequency was below 0.15 and/or multiple alterations were found within the same pattern of NOTCH1 activation (LIA or ID). All patients were received Berlin-Frankfurt-Münster based chemotherapies with non-minimal residual disease (MRD) based risk stratification, which were mainly offered from the Tokyo Children's Cancer Study Group (TCCSG) and the Japan Association of Childhood Leukemia Study (JACLS).ResultsIn total, we detected aberrations activating NOTCH1 signaling in 81.3% (143/176) of cases including subclonal mutations. Subclonal alterations were observed in 26.7% (n=47). Single nucleotide variations in the heterodimerization domain (HD-SNV) were the most frequent (43.2%; n=76), followed by PEST domain mutations (33.0%; n=58), FBXW7 mutations (26.1%; n=46), non-frameshift indels of NOTCH1 (19.9%; n=35), and in-frame internal duplication known as juxta-membrane expansion (6.3%; n=11). Amplification of NOTCH1 region and 5' NOTCH1 deletion were not detected in our cohort. Both LIA and ID patterns were detected in 43.2% (n=76). Most mutations were mutually exclusive within each LIA and ID pattern.Intriguingly, we detected four (2.3%) internal deletion of NOTCH1 gene (DEL; missing exon 3-27 (DEL3) or 21-27 (DEL21)), three cases (1.7%) of SNV at 3' untranslated region, and two (1.1%) SEC16A-NOTCH1 fusions. These alterations were previously reported to activate NOTCH1 signaling in breast cancer or chronic lymphoblastic leukemia, except for DEL21. We confirmed that DEL21 strongly activates NOTCH1 signaling by luciferase reporter assay (over 100 times compared to wild type NOTCH1). As previously reported in DEL3 and CUTLL cell line, transcripts might initiate at methionine 1737 located within the NOTCH1 transmembrane domain and seem to be sensitive to γ-secretase inhibitors.Analysis of frequency of detected NOTCH1 activating alterations in each previously reported WTS-based cluster (ETP, SPI1, TLX, TAL1-RA, and TAL1-RB) revealed that alterations were frequently detected in TLX (100%; 24/24) and TAL1-RB (95.1%; 39/41), whereas less frequent in TAL1-RA (61.1%; 11/18). In TAL1-RA, all SEC16A-NOTCH1 fusions were observed despite significantly low rate of HD-SNV (11.1%; 2/18). In SPI1 cluster, PEST domain alterations were frequently detected (71.4%; 5/7). Importantly, cases harboring subclonal NOTCH1/FBXW7 alterations showed significantly worse outcome (log-rank P = 0.01), although there was no prognostic difference between cases with and without NOTCH1/FBXW7 mutations.ConclusionsWe observed NOTCH1 activating alterations in 81.3% of pediatric T-ALL cases and detected rare internal deletion of NOTCH1 gene and NOTCH1 fusions recurrently in T-ALL. Furthermore, the presence of subclonal NOTCH1/FBXW7 mutations might be relevant to unfavorable outcome. Despite several limitations such as non-MRD based treatment, our results might be useful for developing a new anti-NOTCH1 therapeutic strategy for pediatric T-ALL patients. DisclosuresNo relevant conflicts of interest to declare.

The Prognostic Value of TP53 Mutations Depends on Clinical Backgrounds in Pediatric Patients with Acute Lymphoblastic Leukemia

IntroductionTP53 mutations in relapsed cases with pediatric acute lymphoblastic leukemia have been implicated in poor clinical outcomes. However, the prevalence and clinical significance of TP53 mutations at diagnosis have not been fully investigated. Such knowledge is essential for the care of patients, because treatment intensity is tailored to predictive prognosis, where increased attention has been directed toward de-escalation of treatment for the problem of long term effects and second malignancies in childhood cancer survivors.MethodsMutation status of TP53 was detected by targeted-capture sequencing of TP53 coding regions in 1,003 children with B-precursor ALL who had been treated in either of the two prospective clinical trials, JACLS (Japan Association of Childhood Leukemia Study) ALL-02 and TCCSG (Tokyo Children's Cancer Study Group) L04-16. Detection of common fusion genes, including BCR-ABL, ETV6-RUNX1, MLL-AF4, MLL-ENL, MLL-AF9, and TCF3-PBX1, were performed using qPCR assays. We designed SNP baits to analyze copy number status of chromosome 17, and also captured 662 probes tiling the entire IgH enhancer locus to identify IGH-DUX4 rearrangement.ResultIn total, 36 different non-silent coding TP53 mutations were identified in 30 (3%) patients, including 22 missense, 7 frameshift indel, 5 in-frame indel, and 2 nonsense mutations. All missense mutations were found in the core DNA-binding domain (n=21), except for one mutation, which affected the tetramerization motif. Variant allele frequencies (VAF) of TP53 mutations varied from 3% to 97% with 14 mutations showing < 10% VAFs. Showing a significant correlation with mutated TP53 (Odds ratio 20: 95%CI 6.4-61, P<0.001), loss of heterozygosity affecting the TP53 locus was observed in 11 (37%) cases and caused by del(17p) in most cases (n=10). We next evaluated clinical features of TP53-mutated cases. TP53 was most frequently mutated in Hypodiploid ALL (33% n=3), followed by MLL rearrangement (12% n=4), IGH-DUX4 (9% n=5), Others (3% n=8), TCF3-PBX1 (2% n=2), Hyperdiploid (2% n=6), and ETV6-RUNX1 (n=2 0.9%). TP53 mutations were not associated with age or white blood cell count at diagnosis. However, significantly more patients were categorized into National Cancer Institute (NCI) high risk (HR) category (Odds ratio 2.4: 95%CI 1.1-5.3, P = 0.03) and TP53 mutation was associated with a significantly shorter overall survival (OS) among NCI-HR patients (n = 16; HR for death, 6.3; 95% CI, 3.1-13; P<0.001). Five-year OS of NCI-HR patients with TP53 mutations was 44%, suggesting that early treatment intensification or alternative treatment strategies are warranted for these patients. TP53 mutations were also associated with a shorter OS in MLL rearrangement and IGH-DUX4 ALL. Particularly, 67% (n=4/6) of cases with any cause of death harbored TP53 mutation in IGH-DUX4 ALL. In contrast, TP53 mutation was not associated with shorter overall survival in NCI-SR cases. In Hyperdiploid ALL, 5 out of 6 cases with TP53 mutations were categorized into the NCI-SR category and were all alive. Prognostic impact of TP53 mutation was also investigated using recursive partitioning to generate a hierarchical prognostic model for OS by incorporating genetic subgroups and the NCI risk criteria. This model also demonstrated that the NCI risk criteria was the most important prognostic variable and TP53 mutation was used for stratification of patients only in the NCI-HR category.ConclusionTP53 mutations at diagnosis are common in Hypodiploid ALL and also found in a substantial fraction of MLL rearrangement and IGH-DUX4 ALL, where the mutations predict a poor prognosis. TP53 mutation is also found in NCI-SR cases but may not be associated with poor prognosis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Low Incidence of Osteonecrosis in Childhood Acute Lymphoblastic Leukemia Treated With ALL-97 and ALL-02 Study of Japan Association of Childhood Leukemia Study Group.

Purpose Osteonecrosis (ON) is a serious complication of the treatment of childhood acute lymphoblastic leukemia (ALL); however, data relating to ON in Asian pediatric patients with ALL are scarce. Therefore, we performed a retrospective analysis of cohorts of Japanese patients with ALL to clarify the incidence, clinical characteristics, and risk factors of ON. Patients and Methods The incidence and characteristics of ON were determined in patients with ALL (n = 1,662) enrolled in two studies from the Japan Association of Childhood Leukemia Study (JACLS) group (n = 635 and n = 1,027 patients treated with the ALL-97 and ALL-02 protocols, respectively). Results In total, 24 of 1,662 patients suffered from ON, of which 12 of 635 and 12 of 1,027 patients were treated with the ALL-97 and the ALL-02 protocol, respectively. Of the 24 patients, 23 were older than 10 years. In multivariate analysis, age (≥ 10 years) was the sole significant risk factor for ON ( P < .001). Separate evaluation of patients ≥ 10 years of age indicated a 5-year cumulative incidence of ON of 7.2% (95% CI, 4.0% to 12.6%) and 5.9% (95% CI, 3.3% to 10.4%) in the ALL-97 and the ALL-02 protocol, respectively, which was lower than reported previously, despite an administration of dexamethasone (DEX) similar to that in comparable studies; however, concomitant administration of DEX and l-asparaginase was reduced in the JACLS protocols. Conclusion We identified a low frequency of ON in the JACLS ALL-97 and ALL-02 studies. Although the sole risk factor for ON was age (≥ 10 years), even among high-risk patients, ON incidence was significantly lower than that reported in previous studies. These results suggest that, not only the total amount of DEX, but also how DEX and l-asparaginase are administered, which affects the clearance of DEX, may be associated with ON incidence in patients with ALL.

The Outcome of Low-Risk Childhood B-Cell Precursor ALL Treated with the Japan Association of Childhood Leukemia Study (JACLS) ALL-02 Trial

Background: It is important to establish proper intensity of chemotherapeutic regimen for low risk group of childhood B-precursor ALL (BCP-ALL) to avoid late effects. For this purpose, in Japan Association of Childhood Leukemia study (JACLS) ALL-02 clinical trial, we defined standard risk (SR) group as the patients ranged from 1 to 10 years showing white blood cell (WBC) count of less than 10,000/μL at the diagnosis without predonisolone poor responder, t(4;11), t(1;19) and CNS3 to treat with less intensive chemotherapy (JACLS ALL02 SR protocol). Herein, we report the outcome of JACLS ALL02 SR protocol and analyzed prognostic factors to identify super low risk group which is curable by less intensive chemotherapy.Patientss and treatment: JACLS ALL02 SR protocol consisted of early phase therapies for total 4 months including an induction therapy using vincristine (VCR), steroids, pirarubicin (THP-ADR), and L-asparaginase (L-asp) for 4 weeks, a consolidation therapy using cyclophosphamide and a combined administration of cytarabine and dexamethasone (DEX) or 6-mercaptopurine (6-MP), a sanctuary therapy with only two cycles of high-dose methotrexate (MTX) of 3g/m2, one course of 4 drugs re-induction therapy and maintenance phase with 6-MP and oral MTX combined with VCR and PSL every 5 weeks by the end of the second year. In addition, total of 12 times of triple intrathecal therapy were administered by the end of the first year. Estimation of event free survival (EFS) and overall survival (OS) was performed using the Kaplan-Meier method and the differences were compared using the log-rank test. Multivariate analysis was performed using a Cox regression model.Results: 1252 newly diagnosed ALL patients were enrolled from April 2002 to May 2008 in JACLS ALL02 clinical trial, which include 388 patients (192 males and 196 females) in SR group (31.0%). The median age at diagnosis was 4.2 years (range, 1.0 to 9.9 years) and the median WBC count was 3,700/μL (range, 370 to 9,984 /μL). The median follow-up time was 6 year and 5 months. 4y / 8y EFS and OS (±SE) of the SR group are 91.5±1.4% / 89.4±1.7% and 97.9±0.7% / 95.2±1.2%, respectively. 4y / 8y EFS of the patients of 1 to 5 years old (n=300) and 6 to 9 years old (n=88) are 93.5±1.4% / 93.0±1.5% and 84.9±3.9% / 75.8±5.8%, respectively (log rank p=0.0003). 4y / 8y EFS of the patients with day 15 M1 marrow (n=287 ) was 92.8±1.6% / 91.6±1.8%, while those of the patients with day 15 M2 marrow (n=101) was 88.0±3.3% / 83.7±3.9% (log rank p=0.0446). When 388 patients were further classified into 4 subgroups based on genetic abnormalities, such as ETV6-RUNX1 (n=89, 22.9%), high hyperdiploid (HHD) (n=97, 25%), normal karyotype (n=147, 37.9%), and others (n=55, 14.2%), 4y-EFS/OS of each group was 97.6±5.9 /100% (ETV6-RUNX1), 91.5±2.9 /100% (HHD), 89.6±2.5 /95.1±1.8% (normal karyotype), and 86.9±4.6 /98.2±1.8% (others), suggesting poor EFS of B-others group. When HHD subgroup was divided into two groups, such as triple trisomy (TT; n=35) (trisomy of chromosome 4,10,17) and non-TT (n=62), 4y-EFS of each sub-group was 100% and 84.5±4.5%, respectively (log rank p=0.0161). Interestingly, day 15 M2 marrow was associated with poor 4y/8y EFS only in the normal karyotype subgroup (day 15 M1 vs M2 92.0±2.6% / 90.2±3.1% vs, 81.3±6.9% / 77.2±7.7%, log rank p=0.0497). The rates for NCI-CTC grade 4 infection, allergy, increase of transaminase were 1.80%, 0.26%, and 22.9%, respectively. Multivariate analysis identifies the patients of 6 to 9 years old (HR=3.178, p=<0.001) as a predictor of poor prognosis in EFS and day 15 M2 marrow (HR=3.273, p=0.027) and B-others subgroup (HR=11.1, p=0.0017) as predictors of poor prognosis in OS.Discussion: Although JACLS ALL-02 SR protocol was less intensive chemotherapeutic regimen compared to the contemporary protocols used for NCI-SR patients, good outcome of the 4y EFS and OS (91.5±1.4 % and 97.9±0.7 %) was achieved with good feasibility. Because the specific genetic subgroups such as B-others and HHD without TT were associated with relatively poor outcome, those patients should be treated with more intensive chemotherapy, especially for the patients showing initial poor response (day 15 M2). On the other hand, ETV6-RNUX1 and TT subgroups showed excellent outcome. Thus, appropriate reduction of therapy should be explored for these subgroups. DisclosuresNo relevant conflicts of interest to declare.

Shift to Anti-Fibrinolysis on the Administration of L-Asparaginase (L-Asp) during Induction Therapy for Pediatric Acute Lymphoblastic Leukemia (ALL)

Introduction: Thromboembolism is a serious complication associated with ALL. The use of central venous catheter and treatment protocols involving corticosteroids and L-Asp is assumed as important thrombogenic factors at the induction phase. In particular, L-Asp has profound effects on hepatic synthesis of pro-, anti-coagulant and fibrinolytic factors. In this study, we hypothesized that change of coagulation and fibrinolytic function contributes to hyper-coagulation condition during the induction phase with L-Asp. In order to clarify this, we evaluated the dynamic change in coagulation and fibrinolysis by simultaneous measurement of both thrombin and plasmin generation assay (T/P-GA). Patients: Twenty-seven pediatric patients with newly diagnosed ALL were enrolled from Aug. 2014 to Oct. 2015 at 3 hospitals in Japan. All cases had no thrombotic predisposition. Eighteen cases (66.7%) (BCP-ALL; n=17, T-ALL; n=1) received Berlin-Frankfürt-Münster (BFM)-95 oriented induction therapy included prednisolone (and dexamethasone for T-ALL), vincristine, daunorubicin, and E.coli L-Asp (a total of 8 doses of 5,000 U/m2). The others (BCP-ALL; n=8, T-ALL; n=1) received Japan Association of childhood Leukemia Study (JACLS) ALL02 oriented induction therapy included prednisolone, dexamethasone, vincristine, daunorubicin, cyclophosphamide and E.coli L-Asp (a total of 6 doses of 6,000 U/m2). Methods: The individual hemostatic parameters were monitored by fibrinogen (Fbg), FDP, AT, TAT and PIC. Additionally, the global functions of coagulation and fibrinolysis were evaluated using T/P-GA established by our group [Matsumoto et al. TH 2013]. This assay was initiated by the addition of a mixture of optimized concentrations of tissue factor and tissue-type plasminogen activator. Thrombin and plasmin generation were monitored simultaneously using individual fluorescent substrates in separate microtiter wells. Standard curves were set using purified alpha-thrombin and plasmin. Patients' plasmas were collected at the following points, T0; pre-phase of L-Asp, T1; intermittent phase of L-Asp, T2; post-phase of L-Asp, and T3; post-induction phase. Endogenous potentials of thrombin generation (T-EP) for coagulant activity and plasmin peak levels (P-Peak) of plasmin generation for fibrinolytic activity were selected as parameters for evaluation in this study. A ratio of T-EP and P-Peak of patients' plasmas to those of control normal plasma were calculated. Results: All cases obtained first remission, and none of them developed coagulopathy. Six cases received FFP transfusion for low Fbg level, whilst 21 cases received AT supplement for low AT level. Fbg showed a median of 170, 99.0, 99.0 and 328 mg/dl at T0, T1, T2 and T3, respectively, whilst the other individual parameters showed relatively unchanged. T-EP revealed a median of 1,126, 1,059, 1,175, 1,343 and 1,132 nM, whilst P-Peak showed a median of 6.67, 4.54, 4.12, 5.50 and 5.77 nM for T0, T1, T2, T3 and control plasma, respectively, indicating the elevated T-EP ratios and reduced P-Peak ratios (Fig. 1). The most significant difference in both ratios demonstrated a median of 1.5-fold (range, 1.0 to 2.6) at T2, consistent with the lowest Fbg levels. The FFP transfusion group showed significantly lower T-EP ratios than non-transfusion group at T1 (a median of 0.87 vs. 1.01, P=0.041) and T2 (a median of 0.96 vs. 1.07, P=0.009), whilst P-Peak ratios revealed no significant changes. The AT supplement group showed no significant changes of both ratios. Conclusion: The results from decreased Fbg and unchanged FDP might reveal the hepatic synthesis disorder of Fbg, whilst the results from T/P-GA showed that their hemostatic dynamics appear likely to be thrombotic tendency, since their coagulation state was hyper-coagulation and anti-fibrinolysis at post-phase of L-Asp. These results suggest that the impaired balance of coagulation and fibrinolysis due to L-Asp therapy might play an important role of a thrombotic complication at induction phase. On the other hand both conventional FFP transfusion and AT supplement therapy might not dramatically repair this unbalance state. A further research would be required to examine the role of coagulant and fibrinolytic function using T/P-GA in the pathogenesis of coagulopathy associated with L-Asp therapy in order to establish the optimal supportive therapy. [Display omitted] DisclosuresNogami:F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Matsumoto:Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties, Research Funding. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding.

Identifications of Highly Aggressive Phenotype with SPI1 Overexpression in Pediatric T Cell Acute Lymphoblastic Leukemia/Lymphoma

T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) accounts for 10% to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia (ALL), arising from the malignant transformation of hematopoietic progenitors primed toward T cell development, as result of a multistep oncogenic process. However, since the prognostic significance of these genetic alterations in pediatric T-ALL is not clear, genetic basis which contributes aggressive phenotype or progression of pediatric T-ALL is still to be elucidated. Therefore, to discover driver genetic events, which involved in the aggressive phenotype of pediatric T-ALL and to identify it's novel prognostic markers, we performed integrated genetic analysis in a large cohort of T-ALL case.Our cohorts included samples from Tokyo Children's Cancer Study Group (TCCSG) and Japan Association of Childhood Leukemia Study (JACLS). Whole transcriptome sequencing (WTS) was performed in 123 cases. Representative recurrent fusion genes were as follows, SIL-TAL1 (n=25), MLL-ENL (n=5), PICALM-MLLT10 (n=5), and NUP214-ABL1 (n=2). Intriguingly, novel recurrent in-frame SPI1 fusions (STMN1-SPI1 n=2; TCF7-SPI1 n=5) were detected, and RT-PCR analysis in additional 60 cases revealed other 2 TCF7-SPI1 fusions. Thus, SPI1 fusions accounted for 4% of pediatric T-ALL/LBL. Expression data of WTS revealed cases with SPI1 fusion showed significantly higher expression of SPI1 compared to cases without SPI1 fusion, implicating that aberrant high expression of SPI1 involved in leukemogenesis.To address the functional activities of SPI1 fusions, we performed luciferase assay using the reporter vector contained the CSF1 promoter region with SPI1 binding site. Transient transfection of Hela cells with the SPI1 fusions expression vectors as well as the wild type SPI1 expression vector showed strikingly high levels of transcription of the reporter genes, as compared to transfection with the empty expression vector, indicating that both SPI1 fusions have transcriptional activities.Next, to analyze the leukemogenic potential of SPI1 fusions in vitro, we transduced fusions cDNA into mouse double negative T-cells. Since p16(CDKN2A) is frequently silenced in T-ALL, we also used p16 null double negative T-cells. Both wild-type and p16 null double negative T-cells expressing SPI1 fusions showed increased cell proliferation compared to the MOCK cells.We further evaluated the impact of SPI1 fusions on T cells differentiation. TCF7-SPI1 or MOCK vector was transduced mononuclear cells isolated from mouse bone marrow. These cells were cultured under stimulating factors, such as IL6 and TPO for 3 days, and then transplanted into the irradiated mouse. Subsequently, 6 week after transplantation, FACS analysis was performed. Of interest, significantly high population of cells expressing TCF7-SPI1 was observed in the immature single positive stage, implicating that their differentiation was impaired at the pre-T cell stage. These results indicate that novel SPI1 fusions have a potential leukemogenic effect in pediatric T-ALL. We defined SPI1 overexpression cases as outliers of SPI1 expression, resulting in extremely poor prognosis (log-rank p = 1.9 ×10-6). Of note, significant poor outcome was confirmed by univariate and multivariate analysis in cases with SPI1 overexpression cases (log-rank p = 9.3 ×10-6, and p = 2.0 ×10-6, respectively).In conclusion, SPI1 fusions expressing cells expanded and they remained at an immature stage, implicating a potential leukemogenic activity of these fusions. Not only the cases with SPI1 fusions, but also the cases with high SPI1 expression without fusions showed extremely poor prognosis, suggesting the prognostic value of aberrant SPI1 expression in pediatric T-ALL. Although it remains unclear, why cases with SPI1 fusions/high SPI1 expression have a poor prognosis, our results indicate that these cases are genetically distinct subgroup from other pediatric T-ALL. DisclosuresKataoka:Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria. Ogawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Landscape of Driver Mutations and Their Clinical Impacts in Pediatric Acute Lymphoblastic Leukemia

IntroductionB-progenitor acute lymphoblastic leukemia (B-ALLs) accounts for 85% of pediatric ALL and categorized into several molecular subgroups according to their ploidy and recurrent translocations, such as ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, and MLL-rearrangements. In addition, recent genetic studies using high-throughput sequencing have disclosed landscapes of gene alterations in each subgroup, however, their clinical relevance have not fully been investigated in a large cohort of B-ALL patients who are uniformly treated and enrolled in an unbiased manner.MethodsWe enrolled a total of 515 pediatric B-ALL patients, who had been uniformly treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-02 protocol between 2002 and 2008. These patients were categorized into three risk groups, including standard-, high-, and extremely high-risk. Infantile ALL as well as BCR-ABL1-positive and Down syndrome-associated cases were excluded. A total of 158 known or putative driver genes in pediatric ALL were analyzed for somatic mutations by targeted-capture sequencing. IgH rearrangements were captured using 662 baits tiling the entire IgH enhancer locus. Finally, an additional 1205 baits was also designed to enable sequencing-based genome-wide copy number detection.ResultsThe median age at diagnosis and observation period were 5.2 (1-18.5) and 4.2 (1.8-9) years, respectively. Sixty-six of the 515 patients (13%) had relapsed diseases and 47 patients (9%) had been died. Real-time RT-PCR and conventional cytogenetic analyses revealed subgroup-defining genetic lesions in 368/515 (71%) patients: 117 (23%) cases with ETV6-RUNX1, 48 (9%) with TCF3-PBX1, 13 (3%) with MLL rearrangements, together with those with hyper- (169 [33%]), and hypo- (6 [1%]) diploid. Remaining 162 patients (31%) had none of these abnormalities. The mean depth of the targeted sequencing was 569× across the entire cohort. In total, 823 driver mutations (median 1 per patient, range 0-7) and 954 focal deletions (median 2 per patient, range 0-13) were detected in 483 patients (92%). Among these, most frequently detected were mutations/deletions in CDKN2A (24%), ETV6 (21%), NRAS (18%), KRAS (18%), and PAX5 (15%). IgH-rearrangements were detected in 51 patients, including IGH-DUX4 (26 [5.0%]), IGH-EPOR (3 [0.6%]) and IGH-CRLF2(2 [0.3%]).Genetic alterations were enriched in several functional pathways, of which most frequent was epigenetic regulation (53%), followed by B-cell development (47%), RAS signaling (46%) and cell cycle (40%). A number of novel recurrent genetic lesions were also identified, including those in DOT1L and PHF6. DOT1L encode an H3K79 methyltransferase and was inactivated by frameshift/nonsense mutations and/or deletions in 19 cases. Although frequently found in T-ALL, mutations of PHF6 had not previously been reported in B-ALL but were detected in 14 cases in the current cohort and strongly associated with TCF3-PBX1 translocation.Significant positive correlations were also demonstrated for an additional 10 combinations of common genetic lesions, suggesting functional links between these combinations. Thus, ERG deletions were highly associated with IGH-DUX4 rearrangement, while mutations in KRAS, NRAS, and CREBBP were significantly enriched in hyperdiploid cases. ETV6-RUNX1 fusion also showed positive correlations with alterations in ETV6, CDKN1B, ATF7IP, VPREB1, BTG1, and WHSC1. Furthermore, mutually exclusive relationship between ETV6-RUNX1 translocationsand FLT3mutations were also identified.Finally, we analyzed the prognostic impact of driver mutations. In multivariate analysis of the entire cohort, 4 genetic alterations were significantly associated with poor prognosis (HR [95%CI]): IKZF1 mutations/deletions (2.6 [1.5−4.8]), EBF1 deletions (3.0 [1.4−6.5]), KDM6A mutations/deletions (2.8 [1.2−6.5]), and TP53 mutations (2.7 [1.2−5.9]). Additional factors (q < 0.1) were identified in subgroup analyses, including alterations in ETV6 (5.4 [1.2−24]), CDKN1B (7.4 [1.6−33]) and CDKN2A (4.2 [1.4−12]) in ETV6-RUNX1 ALL, KMT2D (5.9 [1.3−26]) in TCF3-PBX1 ALLand TP53 (38 [4.1−364]) in IGH-DUX4ALL.ConclusionsWe revealed the landscape of genetic lesions in pediatric B-ALL including novel targets of recurrent mutations with clinical relevance of common genetic lesions. Our results should help in the better stratification of patients. DisclosuresOgawa:Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding.

Continuous Cytarabine Plus Dexamethasone in Consolidation Phase to Patients with Childhood ALL: Result from Japan Association of Childhood Leukemia Study - JACLS ALL02 Protocol -

<Introduction>Since an excellent outcome was observed in a previous study (OCLSG-94) utilizing continuous cytarabine (CA) infusion in the consolidation phase for the treatment of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), a randomization trial was performed to test the superiority of continuous CA infusion to conventional repeated CA injection as consolidation therapy in Japan Association of Childhood Leukemia Study (JACLS) ALL02 clinical trial.<Patients and Methods>Newly diagnosed children with standard-risk (SR) or high-risk (HR) BCP-ALL excluding patients with Ph+ ALL and infants were included in this study. Patients showing prednisolone good response (PGR) were assigned to SR or HR based on the age at onset and initial white blood cell (WBC) counts (SR: age 1 to 9 and WBC less than 10,000 /µL, HR: remaining patients). Patients achieving complete remission (CR) at the end of induction (day 33) proceeded to the randomization. HR patients with M3 marrow on day 15 were excluded. They were randomly assigned to Arm A (cyclophosphamide 750 mg/m(2) 1hr div: day 1 and 8, CA 75 mg/m(2) 1hr div: day 1-6, 8-13 and 6MP 50 mg/m(2) orally, day 1-14) or Arm B (cyclophosphamide 500 mg/m(2) 1hr div: day 1, 3 and 5, CA 100 mg/m(2) 24hr div: day 1-5 and DEX 5 mg/m(2) 1hr div twice a day, day 1-5). Intrathecal chemotherapy (MTX, HDC and CA) was administered twice in this phase in both arms. In the HR group two doses of pirarubicin (25 mg/m (2)) were added and this phase was repeated after re-induction therapy. Estimation of event free survival (EFS) and overall survival (OS) was performed using the Kaplan-Meier method and the differences were compared using the log-rank test.<Results and Discussion>Between April 2002 and May 2008, 1252 children with newly diagnosed ALL were enrolled to JACLS ALL-02 study. Among these, the number of patients with SR and HR were 388 (arm A=200, arm B=188) and 513 (arm A=251, arm B=257, not assigned=5), respectively. The characteristics of eligible patients between arm A and B were not different statistically (Table). Among SR patients, the 4y-EFS were 89.7 +/- 5.2% and 93.4 +/- 4.7% in Arm A and B, respectively (p=0.70). Among HR patients, the 4y-EFS were 89.7 +/- 5.2% and 90.5 +/- 4.4% in Arm A and B, respectively (p=0.97). Although hematological toxicity was not different, in the next phase (sanctuary phase) following Arm A consolidation therapy the frequencies of hematological grade 3 and 4 adverse effects were higher in both SR and HR (SR: p<0.01 in Hb, WBC, and ANC, p<0.05 in Plt; HR: p<0.01 in Hb, WBC, ANC, and Plt). On the other hand, the frequencies of severe adverse effects of liver dysfunction during consolidation phase were higher in Arm B (SR: p<0.05, HR: p<0.01), although grade 3/4 elevation of AST/ALT were not different.<Conclusions>Consolidation therapy with continuous infusion of CA (Arm B) was not superior to conventional CA regimen. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

小児の好中球減少期発熱患者におけるミカファンギンの薬物動態と安全性：小児白血病研究会（JACLS）からの報告

小児の好中球減少期発熱患者におけるミカファンギンの薬物動態と安全性：小児白血病研究会（JACLS）からの報告

Outcome of Adolescent and Young Adults with Acute Myeloid Leukemia Treated with Pediatric Protocols: A Report from the 3 Japanese Cooperative Studies

BACKGROUND: There have been conflicting results from several large pediatric AML collaborative groups comparing survival of adolescent and young adults (AYAs) to that of younger patients with AML. We conducted a retrospective analysis using data from 3 Japanese pediatric AML studies; AML99 (2000-02), AML9805 (1998-2002), and AML-05 (2006-10), conducted by the Japanese Childhood AML Cooperative Study (consisted of Tokyo Children’s Cancer Study Group (TCCSG), Japan Association of Childhood Leukemia Study (JACLS), and Kyushu Yamaguchi Children’s Cancer Study Group (KYCCSG)), the Japanese Childhood Cancer and Leukemia Study Group (CCLSG), and the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG, the first Japanese national study group), respectively.PATIENTS & METHODS: Among the 782 patients with de novo AML (excluding acute promyelocytic leukemia and Down syndrome), 44 were AYAs (age ≥15 years), 574 were age 2 to 14 years, and 164 were infants (age <2 years) at diagnosis. Because of the underlying differences in biological characteristics, further analysis on event-free survival (EFS), overall survival (OS), cumulative incidence of relapse (RR) and treatment-related mortality (TRM) were compared between AYAs and patients 2 to 14 years old.RESULTS: 29.5% (13/44) of AYAs were t(8;21) (vs. 37.1% of 2 to 14 years old, P=0.315), 38.6% (17/44) were normal karyotype (vs. 22.0%, P=0.011), and 16.2% of the tested cases (6/37) were FLT3-ITD positive (vs. 9.4%, P=0.554). Complete remission (CR) rate after 2 induction courses was similar between AYAs and patients 2 to 14 years old: 88.6% vs. 88.8% (P=0.965). Five-year EFS rate was also similar between the two cohorts: 55.2% vs. 59.2% (P=0.578). However, 5-year OS rate was significantly poorer in AYAs: 54.7% vs 73.8% (P=0.005). Total 19 events were observed among the 44 AYA patients; 4 failures to achieve CR, 12 relapses, and 3 deaths. Fourteen out of the 16 patients with non-CR or relapse eventually died and 8 deaths were treatment-related. In fact, 5-year RR was similar between the 2 age groups (32.7% vs. 30.1%, P=0.819), but TRM was significantly higher in AYAs (33.2% vs. 12.4%, P=0.001). In multivariate analysis, using Cox proportional hazard regression model, age ≥15 years old at diagnosis, absence of low risk cytogenetics (t(8;21) or inv(16)), and no remission after initial induction course were associated with both poorer OS and higher TRM, while high white cell count at diagnosis (≥ 100,000/μL) and presence of high risk cytogenetics (-7, 5q-, t(16;21)(p11:q22)/FUS-ERG, Philadelphia chromosome, and/or FLT3-ITD) were not siginificant.CONCLUSIONS: AYAs with AML had inferior OS, but not EFS, due to higher incidence of TRM especially after failure of initial treatment. DisclosuresNo relevant conflicts of interest to declare.

Clinical Significance of LNK (SH2B3) Expression in Pediatric B Cell Precursor Acute Lymphoblastic Leukemia

Background: In pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL), aberrant JAK-STAT signaling is one of the key leukemogenic mechanisms. Although activating mutations of JAK2 are found in high-risk BCP-ALL patients in western countries, such mutations are rare in Japanese cohorts, led to speculation of other factors contributing abnormal activation of JAK-STAT pathway. The adaptor protein LNK (SH2B3) is one of the negative regulator of JAK-STAT signaling, and its loss of function mutations have been identified in myeloproliferative neoplasms and high-risk BCP-ALL. In addition, the loss of function of LNK has been demonstrated to increase proliferation of B cells in vivo. Based on these findings, we conducted genetic analysis to determine the prognostic impact of LNKin BCP-ALL, and functional analysis to investigate its possible mechanisms.Methods: For genetic analysis, we evaluated diagnostic bone marrow or peripheral blood samples of 164 pediatric BCP-ALL patients treated with the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol along with peripheral blood samples from 9 healthy volunteers. The LNK expression level was determined by qRT-PCR. Deletion of IKZF1 was determined by MLPA, and direct sequencing was employed to detect LNK mutations in patients with IKZF1 deletion. For functional analysis, thrombopoietin (TPO)-dependent BaF3-MPL cells expressing LNK (BaF3-MPL-LNK) was established by retroviral transduction to investigate if LNKexpression levels impact on drug sensitivity for prednisolone (PSL), doxorubicin (DOX) or vincristine (VCR). For growth assay, BaF3-MPL cells with or without LNK were cultured for four days in the presence of TPO and the viable cell number was counted. For drug sensitivity test, BaF3-MPL cells with or without LNK were treated with each drug for 48 hours in the presence of TPO, then the IC50 was calculated. Phospho-specific flow cytometory (Phos-Flow) was performed to measure JAK-STAT activation.Results: The LNK expression levels in pediatric BCP-ALL patients’ samples were significantly lower than in samples from healthy donors (P < 0.01). When analyzing all 164 cases, the expression level of LNK was decreased in relapsed patients but there was no statistical significance (P = 0.067). IKZF1 deletion was found in 25 (15% of all) patients, and in these patients, LNK expression level dose not relate to relapse (P = 0.39). Intriguingly, when patients with known high-risk factor (i.e., IKZF1 deletion or poor response to PSL) were excluded, the expression level of LNK was significantly higher in non-relapsed patients (P < 0.05). In functional assay, we observed inhibition of TPO dependent growth of BaF3-MPL cells by expression of LNK (P < 0.01), consistent with previous reports. Phos-Flow analysis revealed that LNK expression suppressed TPO–induced phosphorylation of STAT5 in BaF3-MPL cells. In drug sensitivity test, we found that IC50 of PSL and DOX were substantially lower in BaF3-MPL-LNK cells from in BaF3-MPL-mock cells (0.70 vs 3.93 nM, P < 0.01 and 0.61 vs 1.14 nM, P < 0.05, respectively). Decline in IC50 of VCR by LNK expression was not statistically significant (1.38 vs 2.45 nM, P = 0.056). We next compared the impact of LNK with Ruxolitinib (RUX), a potent synthesized JAK2 inhibitor. The diminution in IC50 of PSL in BaF3-MPL-mock cells treated with RUX (50 nM) was comparable of that in BaF3-MPL-LNK cells (0.31 nM, combination index (CI) = 0.39), consistent with our hypothesis that LNK is working as a JAK2 inhibitor. Since we identified two amino-acid substitutions in N-terminal proline-rich dimerization domain (R139H) and PH domain (P242S), we also examined their function. Transductions of these genes in BaF3-MPL cells, however, did not alter cell growth, suggesting they are single nucleotide variants.Discussions: Our findings that high LNK expression is associated with low relapse rate in intermediate risk (IKZF1 intact, good PSL response) patients indicate potential of LNK to restrain relapse in such patients, presumably by suppressing JAK-STAT signaling. Since we proved the impact of LNK expression to improve sensitivity of PSL in vitro which was comparable to RUX, RUX could compensate lack of internal LNK expression to induce cell death of BCP-ALL cells. Collectively, targeting JAK-STAT pathway will be promising therapeutic option for intermediate risk BCP-ALL patients with low expression level of LNK. DisclosuresNo relevant conflicts of interest to declare.

Outcome of TCF3‐PBX1 positive pediatric acute lymphoblastic leukemia patients in Japan: a collaborative study of Japan Association of Childhood Leukemia Study (JACLS) and Children's Cancer and Leukemia Study Group (CCLSG)

This study reviewed the clinical characteristics of 112 pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients with TCF3-PBX1 fusion treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL02 protocol (n = 82) and Children's Cancer and Leukemia Study Group (CCLSG) ALL 2004 protocol (n = 30). The 3-year event-free survival (EFS) and overall survival (OS) rates were 85.4 ± 3.9% and 89.0 ± 3.5% in JACLS cohort, and the 5-year EFS and OS were 82.8 ± 7.0% and 86.3 ± 6.4% in CCLSG cohort, respectively, which are comparable to those reported in western countries. Conventional prognostic factors such as age at onset, initial white blood cell count, and National Cancer Institute risk have also no impact on OS in both cohorts. Surprisingly, the pattern of relapse in JACLS cohort, 9 of 82 patients, was unique: eight of nine patients relapsed during the maintenance phase and one patient had primary induction failure. However, bone marrow status and assessment of minimal residual disease on days 15 and 33 did not identify those patients. Interestingly, the two patients with IKZF1 deletion eventually relapsed in JACLS cohort, as did one patient in CCLSG cohort. International collaborative study of larger cohort is warranted to clarify the impact of the IKZF1 deletion on the poor outcome of TCF3-PBX1 positive BCP-ALL.

Prognostic Significance Of CRLF2 Over-Expression In Pediatric Acute Lymphoblastic Leukemia

▪ BackgroundPrevious studies have reported that IKZF1 deletion was an adverse prognostic factor in pediatric B-cell-precursor ALL (BCP-ALL). However, the prognostic significance of CRLF2 over-expression (OE) is controversial. In order to assess the prognostic value of CRLF2 OE and genetic alterations involving CRLF2, we conducted genetic analysis of CRLF2 in pediatric BCP-ALL treated according to Japan Association of Childhood Leukemia Study (JACLS) ALL02 cohort. MethodsWe examined diagnostic bone marrow or peripheral blood samples of 167 pediatric BCP-ALL patients treated on the JACLS ALL02 protocol. The analyzed cohort included 68 patients classified in NCI-SR and 99 patients in NCI-HR. The deletion of IKZF1 and gain of CRLF2 were determined by MLPA. CRLF2 expression level was determined by quantitative RT-PCR and OE was defined as 10 times more than mean expression level of 167 cases. The P2RY8-CRLF2 fusion was detected by RT-PCR. To identify fusion transcript related to CRLF2 OE, we performed messenger RNA sequencing (mRNA-seq) on six of 17 patients with CRLF2 OE without CRLF2 gain and P2RY8-CRLF2 fusion. ResultsCRLF2 OE was identified in 30 (18%) of 167 patients, which was similar to that reported previously. IKZF1 deletion was found in 25(15%) of 167 patients, which was found more in CRLF2 OE patients than non-CRLF2 OE patients (30% vs 11%, p<0.05). P2RY8-CRLF2 fusion was identified in only 3 (1.7%) of 167 patients, and all of them were classified in CRLF2 OE. In detail, only one of three P2RY8-CRLF2 positive patients had IKZF1 deletion. One of two P2RY8-CRLF2 positive patients without IKZF1 deletion harbored MLL-AF4 fusion. CRLF2 gain was identified in 18(11%) of 167 patients. Eleven of these 18 patients were classified in CRLF2 OE, suggesting CRLF2 gain was significantly related to CRLF2 OE (37% vs 5%, p<0.01). Interestingly, none of CRLF2 OE patients with CRLF2 gain did have IKZF1 deletion. We identified one patient with novel fusion transcript caused by the 32kb deletion from CRLF2 intron 1. This patient was classified in CRLF2 OE with IKZF1 deletion, suggesting that the novel fusion transcript was related to CRLF2 OE. However, we could not identify any fusion transcripts related to CRLF2, such as IgH@-CRLF2, in remaining five patients by mRNA-seq. Interestingly, we identified three EBF1-PDGFRb positive patients in CRLF2 OE patients with IKZF1 deletion who did not have genetic alterations including CRLF2. In survival analysis, significant difference on the 5-year event-free survival (EFS) and overall survival (OS) between patients with and without CRLF2 OE was not observed (71% vs 75%, log rank p=0.68, 96% vs 82%, log rank p=0.11). In addition, type of genetic alterations related to CRLF2, such as CRLF2 gain and P2RY8-CRLF2 fusion, did not show the impact on EFS and OS in CRLF2 OE patients. However, significant difference on 5-year EFS between CRLF2 OE patients with and without IKZF1deletion was observed (44% vs 83%, log rank p=0.02). In multivariate analysis of 167 patients, only IKZF1 deletion was significant predictors of inferior OS (Hazard ratio: 2.427, 95%CI:1.037-5.679, p=0.04). DiscussionsThe current analysis revealed that CRLF2 OE was caused by several different mechanisms, such as CRLF2 gain, P2RY8-CRLF2 fusion and rare fusion transcript related to CRLF2. However, regardless of type of genetic alterations of CRLF2, concomitant IKZF1 deletion holds the major impact on poor prognosis in CRLF2 OE patients. Especially, CRLF2 gain and IKZF1 deletion were mutually exclusive. Thus, CRLF2 OE caused by CRLF2 gain was not poor prognostic factor for high risk BCP-ALL. The prognostic significance of CRLF2 should be evaluated in consideration of IKZF1 status. Disclosures:No relevant conflicts of interest to declare.